The introduction of RNA sequencing (RNA-seq), as a direct result of rapid progression of next-generation sequencing technologies, has revolutionized the world of transcriptomics. It enables quantification of the complete set of RNA with all its isoforms in a given cell, in far greater accuracy than before. Here, we provide a step-by-step guide to the sample preparation in RNA-seq and an overview of the bioinformatic analysis that is required following the cDNA sequencing.